Transformation with ''E. coli''

From bio-physics-wiki

Jump to: navigation, search

Transform Cells

do before start for one strain

  1. 2 x Prechill 1mm electroporation cuvette on ice
  2. 2 x Eppis
  3. Set electroporation settings to 1.80kV; $25 \mu F$; $200\Omega$;
  4. Prewarm 1ml LB Medium
  5. Prepare agar plates with 25-30$\mu g/ml$ Chloramphenicol (CHL)

Electroporation

  1. Work on ice; add purified vector DNA from Miniprep ($\sim 1\mu g$ of DNA in a volume of $1-6\mu l$) to a $70\mu l$ aliquot of electrocompetent cells; transfer to electroporation cuvette.
  2. Use one aliquot of electrocompetent cells as negative control.
  3. Prepare two eppis with 1ml prewarmed LB.
  4. Electroporate with 1.80kV; $25 \mu F$; $200\Omega$; $\tau = 3-4$ms.
  5. Recover cells immediately in 1ml prewarmed LB; incubate for 1hr at 37°C.
  6. Plate $100 \mu l$ on CHL$^{30}$ LB agar plates with beads; Spread a diluted sample (10$\mu l$ cell suspension 90$\mu l$ centrifuge 5min at 8000 x g; remove 800$\mu l$ supernatant; resuspend in remaining LB; Spread on agar plate; make a negative control with the concentrated sample.
  7. Optionally: check the plasmid by sequencing

This protocol was created after reviewing several protocols. The reviewed protocols were obtained from

http://www.its.caltech.edu/~bjorker/Prep_of_electocomp_cells.pdf
Molecular Cloning: A Laboratory Manual by Joseph Sambrook, David William Russell, CSHL Press
Protocols from other groups at IST Austria